NIH NCRR funds are requested in this resubmission to purchase a 5-laser 2-camera Amnis ImageStreamX with three (20x, 40x, 60x) objective lenses. This new platform is a vastly improved, refined and far more powerful version of the original instrument Amnis introduced approximately eleven years ago. This institutionally supported and expertly supervised instrument will be placed in the principal investigator's existing Department of Pathology Flow Cytometry Core which has been in successful operation for almost 30 years. The requested instrument will serve to fill the gap between the highly detailed (but far more slowly acquired) cellular images obtained by confocal microscopy and the imageless but rapidly acquired data obtained by immunofluorescence-based multiparameter flow cytometry. The ImageStreamX combines these two later technologies, providing a synergistic advantage over each used in tandem. The requested instrument quantifies both the intensity and intracellular location of fluorescent cellular probes and can image at high rates of speed (up to ~2000 cells/sec), allowing our consortium of Massachusetts General Hospital (MGH) investigators to analyze rare sub- populations and highly heterogeneous samples with statistically robust and objective results. This technology will permit some investigations that would otherwise be impossible to perform, to actually be implemented. By combining the speed, sensitivity, and immunophenotyping abilities and capacities of flow cytometry with the detailed imagery and functional insights of microscopy, the requested instrument will be an invaluable addition to represented researchers at both downtown Boston and Charlestown research campuses of MGH, and throughout the greater Partners Health Care System. The instrument utilizes the same dyes and markers employed in fluorescence microscopy and flow cytometry and can perform virtually any standard flow cytometry assay with the added value of quantitative visual inspection and resulting confirmation. The requested instrument will enable 30 investigators representing 77 NIH funded grants to enter into entirely new and useful areas of investigation. There is currently no similar instrumentation at MGH, anywhere in Partners Health Care/Harvard University, or the surrounding geographic region. A wide variety of studies related to cell biology, immunology, autoimmune, infectious disease, oncology and stem cell research will benefit from the placement of this instrumentation in the principal investigator's laboratory. An ImageStreamX demo-unit was placed in the principal investigator's laboratory in February 2010 for one week; pilot data produced from those 5-days is presented in this submission as well as updated data for this 2011 re-submission. The co-investigators in this application represent over 27 million dollars of NIH funded work based at MGH; the entire hospital of approximately 1000 principal investigator's has a NIH budget of approximately $325 million dollars.